This proposal addresses two fundamental but poorly understood areas of the biology and biochmeistry of microtubules and microtubule proteins: the physiological regulation of microtubule assembly and the existence and consequence of direct interaction between membranes and the principal protein subunitg of microtubules, tubulin. In previous studies of microtubule assembly/disassembly, we showed that an increase in pH raises the critical concentration for assembly and increases the drug sensitivity of microtubule in vitro. Our goal now is to determine the role of pH in in vivo microtubule regulation. Using new spectroscopic methods for determination of intracellular pH from fluorescent probes, we will establish the relationship of microtubule disassembly and pH under physiological conditions in the J774 mouse macrophage, determine the role of Na+ - H+ exchange in alkalinization, and extend the analysis to a neuroblastoma line in which spontaneous microtubule disassembly/assembly can be correlated with pH in single cells. The membrane-tubulin studies derive from our observations that tubulin binds to unilamellar phospholipid (PC) vesicles, and causes disruption of their structure by a process that is inhibitable by sub-stoichiometric concentrations of antimicrotubule drugs. We will extend observations on the segmental motion of tubulin, both free and bound, to liposomes by time-resolved fluorescence depolarization methods. We will also determine more precisely the requirements for binding to lipid bilayers and the effects of tubulin on lipid mobility. Rotational mobility will be examined by fluorescence depolarization. Lateral mobility will be determined by excimer spectrofluorometric techniques. We will determine if tubulin induces membrane fusion, and we will attempt to differentiate soluble from membrane-tubulin by selective proteolysis and monoclonal antibodies directed against antigenic determinants that may be affected by binding or are unique to the conformationally altered bound tubulin. These studies should provide new insight into the physiological regulation of microtubule assembly and on the contribution of membrane bound tubulin to the regulation of surface function.